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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
  • C12Q1/708—Specific hybridization probes for papilloma
• • G—PHYSICS
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  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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  • G01N33/56983—Viruses
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C12N2710/00011—Details
  • C12N2710/20011—Papillomaviridae
  • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • G01N2333/01—DNA viruses
  • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/975—Kit
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/811—Test for named disease, body condition or organ function
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/811—Test for named disease, body condition or organ function
  • Y10S436/813—Cancer

Definitions

• the invention relates to papillomavirus DNAs, and more particularly to probes derived from these papillomaviruses, as well as methods using them for the in vitro diagnosis of papillomavirus infections.
• papillomavirus covers a large number of viruses having in common to be held responsible for several forms of viral infections spreading between skin warts or relatively benign mucous membranes and hyperplasias liable to degenerate into intra-neoplasias. epithelial and skin cancer.
• papillomavirus infections mention will also be made more particularly of wart-shaped epidermodysplasia, which will sometimes be referred to hereinafter as "EV”.
• EV wart-shaped epidermodysplasia
• HPV HPV after molecular cloning of their genomes
• KREMSDORF D. et al, 1982, J. Virol. 43: 436-447 and KREMSDORF et al, 1983, J. Virol. 48: 340- 351
• HPVs were divided into three groups based on the absence of cross hybridization or very weak cross hybridization between genomes belonging to different groups.
• the first group included HPV3a and 10 which are associated with the flat warts seen in some EV patients and in the general population; DNA sequences related to those of HPV3a have been found in cancer of patients with EV.
• the second group included HPV5, 8 and 12, the genomes of HPV5 and 8 having been detected in cancers of patients with EV.
• the third group to date consists of a single virus, HPV9. With the exception of a recipient of a renal allograft with immunosuppression, who had been found to be infected with HPV5, the viruses of the last two groups had only been detected in patients with EV, most of them being infected by several viruses. It should be noted that among the 14 types of HPV currently mentioned in the literature (Bibliographic references 1-5, 8.9, 13, 14, 16, 18-20 indicated below), four were found to be associated specifically with EV which is a rare disease.
• the invention provides improved techniques for the identification of papillomaviruses, for example obtained from lesions or biopsy sections and makes it possible to make more precise diagnoses, which may also result in improved prognoses as to the possible evolution. of the lesions involved.
• the papillomaviruses although very different from one another, have sizes of the order of 7000-8000 base pairs. In addition, their genomes may nevertheless have certain degrees of homology.
• papillomaviruses several types of papillomavirus will be distinguished, these are distinguished by their percentages of homologies measured under strict or stringent conditions. It will be said that the papillomaviruses which, in the latter conditions, have percentages of homology of less than 50% belong to different types. It will be noted in this regard that the percentages of homology between viruses of different types can even drop to zero under said strict or stringent conditions. Viruses for which we observe, under these strict or stringent conditions, percentages of homology greater than 50% are considered to belong to the same type and form different subtypes within this same type.
• Hybridization tests under non-strict or non-stringent conditions involve the mutual contacting of DNAs originating from two virus isolates under the following conditions described by HEILMAN CA. et al, 1980, J. Virol., 36, 395-407, and CROISSANT et al, 1982, C.R. Acad. Se. Paris, 294. 581-586 (heteroduplex molecules).
• Hybridization tests under strict or stringent conditions involve the mutual contacting of DNAs originating from two virus isolates under the conditions described by KREMSDORF, D. et al. ((1982),
• the papillomaviruses belonging to the same type present hybridizable sequences having substantially identical nucleotide sequences over 80 to 100% of their respective lengths, these homologous sequences can be reduced to 60%, or even less in papillomaviruses of different types.
• the degree of identity or of analogy of the papillomavirus sequences of different types which hybridize mutually under non-strict or non-stringent conditions can obviously be lower than in the case of papillomavirus belonging to the same type.
• the invention therefore relates not only to DNAs capable of being isolated from the various new papillomaviruses which have been isolated and to the probes which may consist of all or part of these DNAs, but also to mixtures or "cocktails" of types. of papillomaviruses capable of being used more effectively for the diagnosis of various categories of infection, or even of the levels of risk which accompany the discovery in a patient of certain papillomaviruses.
• the invention aims to provide means allowing, in the case of infections manifested by verruciform epidermodysplasias, to better appreciate the degree of risk that the latter evolve towards skin cancers.
• DNA-HPV whole genomes of papillomaviruses
• the physical maps give the position of sites of cleavage by various restriction endonucleases.
• the origin of the cards is generally made up of a single clipping site. Distances to origin are expressed as a percentage of genome length.
• One map unit represents 1% of genome length.
• the invention firstly relates more specifically to each of the DNA-HPVs chosen from all of the DNAs which have sizes which range between 7000 and 8000 base pairs and are characterized by the restriction maps which appear in the drawings.
• DNA-HPVs obtained from papillomaviruses which correspond to the designations HPV-2d, HPV-10b, HPV-14a, HPV-14b, HPV-15, HPV-17a, HPV-17b, HPV- 19, HPV-20, HPV-21, HPV-22, HPV-23, HPV-24, HPV-28, HPV-29, HPV-31 and HPV-32, HPV-IP2 and HPV-IP4.
• the invention also extends its effects to DNA-HPVs which can be considered as belonging to the same types as those which have just been stated.
• the physical maps corresponding to the DNA-HPVs of the newly characterized viruses are indicated by a black circle.
• the invention also relates to fragments of the preceding HPV DNAs or capable of hybridizing with these, in particular under strict conditions. Similarly, it relates to the recombinant DNAs containing all or part of each of the DNA-HPVs mentioned above, and more particularly to the recombinant DNAs containing fragments corresponding to the genes E1, E6-E7, L1 and L2 respectively or even fragments containing sequences corresponding to the intergenic regions of said DNA-HPVs. Finally, it relates to the probes which can be formed from these respective DNA-HPVs or from the corresponding fragments and the in vitro diagnostic methods involving said probes.
• the viral DNA preparations were selectively extracted (LUTZNER, MA et al., 1983, Lancet ii: 422424) from scraping products of benign lesions of six European patients with EV and two South American patients with of EV.
• the HPV DNAs were purified by equilibrium centrifugation in cesium chloride gradients and / or sedimentation in sucrose gradients in the presence of ethidium bromide, according to the procedures described above (articles by KREMSDORF, D. and al. already described and ORTH, G. et al., 1980, Cold Spring Harbor Conf. Cell Proliferation 7: 259-282).
• the DNAs were inserted in the form of unit length molecules with the exception of two DNA fragments from HPV24 produced by the endonuclease BamHI. They were inserted either into the plasmid pBR322 (SUTCLIFFE, JG, 1978, Nucleic Acids Res. 5: 2721-2728), using the unique cleavage sites of Aval, BamHI and HindIII, or into a recombinant plasmid having integrated the HindIIIII B fragment of the DNA of HPV5 (article by KREMSDORF, D. et al., 1982, already mentioned), which contains a unique SacI site.
• HPV17b and 22 were inserted in the form of DNA molecules of unit length after cleavage with an enzyme (SacI) which cleaves only once the HPV DNA and the recombinant plasmid pBR322 containing the HindIIIII B fragment HPVS DNA.
• SacI an enzyme which cleaves only once the HPV DNA and the recombinant plasmid pBR322 containing the HindIIIII B fragment HPVS DNA.
• HPV14a DNA was inserted into plasmid pBR322 as a unit length DNA molecule after incomplete digestion of the viral DNA preparation with HindIII, an enzyme that produces two fragments of 96.1 and 3 , 9% of the length of the genome.
• the BamHI A and B fragments of HPV24 (having sizes corresponding respectively to 83.1 and 16.9% of the length of the genome) were inserted separately into the plasmid pBR322.
• the electrophoretic mobilities of the digestion products of the recombinant DNAs and of the uncloned HPV DNAs were compared after treatment with a mixture of two restriction endonucleases comprising the endonuclease used for the insertion of the viral sequences into the plasmid.
• the number and size of the isolated fragments indicated that in each case the whole viral genomes were integrated.
• a heterogeneity of the sizes of the DNAs was observed when the DNAs of the HPVs, not cloned or excised from the plasmid sequences, were analyzed by electrophoresis on agarose gel (data not shown).
• the DNAs of HPV14b, 19, 20 and 21 have sizes similar to those of HPV3a, 5, 8 and 12 (approximately 7700 pairs of nucleotides (articles from KREMSDORF, D. already mentioned), while the DNAs of HPV15, 17a, 17b, 22 and 23 have smaller sizes similar to that of HPV9 (about 7200 nucleotide pairs) (articles by KREMSDORF, D., 1982) and ORTH, G., 1980, already mentioned).
• the sensitivity of the cloned viral genomes to 14 restriction endonucleases was analyzed and the physical maps were established (FIGS. 1 to 10). The restriction maps of some of the DNA-HPVs are repeated in some of the figures for the reasons explained below.
• HPV14a No obvious analogy was detected between these cards and those previously established for HPV associated with EV (HPV3a, 5, 8, 9, 10 and 12) (8,9, 16, 18, 20), to warts skin (HPV1, 2, 4 and 7), and mucocutaneous or mucous membrane lesions (HPV6b, 11a, 13 and 16) (1, 33, 19), with the exception of the closely related HPV14a map à la carte of an HPV isolated from a Japanese patient with EV (24).
• the latter isolate differs from HPV14a by an additional BamHI site and an HindII site, while the locations of the Downstream, BamHI, Bgl1, EcoRI, HindII and HindlII sites are similar in the two viruses. Cross-hybridization experiments confirmed that these two viruses were very closely apparent.
• the non-cleavage enzymes were Pvul, Sal I and Smal for the DNAs of HPV14a and 23; Pvul, Sacl, Sali and Smal for DNA of HPV14b; Bgll, Pvul, Sali and Smal for DNAs of HPV15, 17a and 17b; Bgll, Sacl, Sali and Smal for DNA of HPV19; EcoRI, Pvul , Sacl and Smal for DNA of HPV20; Sac I and Smal for DNA of HPV21; BamHI, Bgll, Pvul, PvuII and Sali for DNA of HPV22; Bgll, EcoRI, Pvul, Sacl, Sali and Smal for HPV24 DNA.
• HPV3a 5, 8, 9, 10 and 12
• HPV1 HPV associated with skin warts
• HPV6b HPV associated with lesions of the mucous membranes
• Hybridization experiments by fixation on a filter paper and DNA-DNA hybridization in phase saturated liquid followed by digestion with nuclease S1 were carried out under strict or stringent conditions previously described (8, 9).
• the HPV DNAs were labeled by nick-translation and fractionated by sedimentation in alkaline sucrose gradients (5 to 20%) as previously described (13).
• the labeled HPV DNAs (4000 cpm) were incubated in 0.48 M NaCl 1 mM EDTA (pH 6.8) at 68 ° C, in the presence of either calf thymus DNA (20 ⁇ g), or d 'Unlabelled HPV DNA (0.20 ⁇ g) as previously described (8, 9).
• the specific activities of HPV DNA probes varied between 5.3 x 10 7 and 2 x 10 8 cpm / ⁇ g.
• the percentage of hybridization was determined by measuring the S1 nuclease-resistant fractions. The numbers represent the values corrected for the spontaneous self-naturalization of the probes (4 to 15%) and normalized to 100% for the homologous hybridization (75 to 95%).
• the abbreviation ND means: not determined.
• the relative importance of cross-hybridizations between DNA-HPV under the conditions indicated above are expressed in% of hybridization between a labeled HPV DNA and an unlabelled HPV DNA.
• HPV14 15, 17, 19, 20, 21, 22, 23 and 24
• HPV17a and b two subtypes of types 14
• HPV17a and b 17
• Table III This table lists the diseases which have been diagnosed in the carriers of these HPVs (isolated or in combination with each other) and the oncogenic potentials which have been recognized for them.
• the DNAs have cross-hybridization rates between them (group homologies) varying from 5 to 38%, and only exhibit notable cross-hybridization (4 to 13%) with the DNAs of HPV5, 8 and 12. These viruses are therefore part of a group of HPV EV previously defined (9).
• HPVs types 13 and 31 can be considered to belong to the same group.
• HPVs of types 1, 2, 4, 24 and 32 which show almost no homology with the genomes of other HPVs are considered to form the first members of other groups distinct from each other and from previous groups
• the invention relates more particularly to DNA fragments, derived from the DNA-HPVs described above, and more particularly those corresponding respectively to the E6-E7 genes; E1; L2; L1 and their intergenic regions.
• the positions and relative lengths of these various fragments, with respect to the sites taken as origins are indicated in Table IV which follows.
• the physical maps of HPV10b, 28 and 29 were aligned with respect to the physical maps of HPV3a and 10a after juxtaposition of the conserved restriction enzyme sites.
• the values of the coordinates given in Table IV indicate the position, on the physical maps presented in figs. 1-9, 5 'and 3' ends of the segments of the genomes homologous to the E6 and E7, E1, L2 and L1 genes and of the intergenic region with respect to the genome of HPV1a or, in the case of HPV31, with respect to the genome of HPV6b.
• the intergenic region (comprising regulatory elements) and the adjacent genes E6 and E7 (presumably corresponding to the major transformation genes expressed in tumors) do not exhibit sequence homology detectable by analysis with an electron microscope of heteroduplex molecules formed, in non-strict hybridization conditions, between genomes of HPV types belonging to different groups, or formed, under strict hybridization conditions, between genomes of most HPV types belonging to the same group.
• the E1 gene (mainly involved in the replication of viral DNA) and the L1 gene (coding for the major protein of the viral capsid carrying the main antigenic determinants of virions) show sequence homologies detectable by heterodu analysis plexes formed, under non-strict hybridization conditions, between genomes of HPV types belonging to different groups or formed, under strict hybridization conditions, between HPV genomes belonging to the same group.
• Probes prepared from recombinant plasmids comprising the E1 and L1 regions can theoretically make it possible to detect the greatest number of types of HPV by molecular hybridization experiments carried out, as the case may be, under strict or non-strict conditions.
• Probes prepared from recombinant plasmids comprising the intergenic region and the E6 and E7 genes make it possible to specifically detect a type of HPV or related types of HPV.
• the L2 region (coding for a minor constituent of the viral capsid) exhibits a variable degree of conservation of nucleotide sequences among the different types of HPV.
• HPV-IP2 and HPV-IP4 viruses were isolated are further described in more detail, then the conditions under which the DNA-HPVs were obtained from these viruses.
• HPV IP2 Molecular cloning and characterization of a new type of HPV associated with neoplasias and genital cancers
• the 8 kb molecules were inserted, via the BglII site, into a vector consisting of the plasmid PL15.5 (containing a single site for cleavage by BglII and by BamHI) inserted by its BamHI site, into the DNA of the bacteriophage lambda L47. 1.
• the lysis plaques corresponding to recombinant phages were detected by hybridization of replicates of the infected bacterial cultures, with a radioactive HPV16 DNA, in non-strict conditions.
• cleavage of phage DNA by the insertion enzyme BglII generates an 8 kb fragment hybridizing with the HPV16 probe under non-strict conditions; cutting the DNA of the recombinant phages and the DNA of the original tumor by mixing the enzymes BglII and PstI generates the same 5 fragments in the sum of the molecular weights is equal to the size of a papillomavirus genome.
• the DNA of the new HPV was excised from the DNA of the recombinant bacteriophages, purified by electroelution, and recloned in the plasmid PL15.5.
• a restriction map of viral DNA was established from the sensitivity of this DNA to 18 restriction endonucleases, which made it possible to locate 21 cleavage sites (FIG. 9).
• the map thus established is different from the map of the HPV genomes identified to date.
• the sequence homology between the DNA of the new HPV and the DNA of the HPVs identified to date has been analyzed by replica molecular hybridization experiments carried out under strict conditions. The homology detected was always less than 5%, the greatest homology being detected with the genome of HPV16.
• the new virus characterized from cervical cancer therefore constitutes a new type of HPV, provisionally called HPVIP2.
• HPVIP2 radioactive probes prepared from the DNA of purified 1 ⁇ PVIP2 made it possible to determine the pathogenic power of these viruses.
• the DNA of HPVIP2 was revealed in one case of bowenoid papules of the external genital organs out of the 14 studied, in 2 invasive cervical cancers out of the 51 studied and in 1 case of intraepithelial neoplasia of the cervix. 'uterus out of the 28 studied, HPVIP2 therefore constitutes a type of HPV with genital tropism presenting an oncogenic potential, the frequency of which is slightly lower than that of HPV18, and much lower than that of HPV16.
• HPV DNA intended for the preparation of molecular probes, for the diagnosis or screening of the types of HPV constituting a risk for the development of genital neoplasias and, in particular, cervical cancer.
• HPV DNA Molecular cleavage and characterization of a new type of HPV associated with precancerous lesions of the skin (HPV
• HPV31 A new type of HPV has been demonstrated in DNA extracted from a biopsy of actinic keratosis, a precancerous skin lesion, by molecular hybridization, under strict conditions, with a mixture with radioactive probes specific for HPV type 5 , 8 and 14. No cross hybridization was detected when the hybridization was carried out with specific probes of types 1, 2, 3, 7, 10, 13, 16, 18, 28, IP1 (previously called HPV31), IP2, and IP3 (previously called HPV32).
• the lysis plaques corresponding to recombinant phages were detected by hybridization of replicates of the infected bacterial cultures, with a radioactive mixture of the DNA of HPVs, 8, and 14, under non-strict conditions.
• a restriction map of viral DNA was established from the sensitivity of this DNA to 15 restriction endonucleases, which made it possible to locate 23 cleavage sites (FIG. 10).
• the map thus established is different from the map of the HPV genomes identified to date.
• the sequence homology between the DNA of the new HPV and the DNA of the HPVs identified to date has been analyzed by replica molecular hybridization experiments carried out under strict conditions. Homology, less than 50%, was detected between the DNA of the new HPV and the DNA of certain types of HPV previously identified in lesions of epidermodysplasia verruciformis (HPVS, 8, 12, 14,19,20, 21 and 25), but no homology was detected with the other types of HPV.
• the new virus characterized from actinic keratosis therefore constitutes a new type of HPV provisionally called HPV-IP4.
• HPVIP4 constitutes a type of skin tropism HPV with oncogenic potential. It is necessary to incorporate it into any mixture of HPV DNA intended for the preparation of molecular probes for the diagnosis or screening of the types of HPV constituting a risk for the development of precancerous or cancerous lesions of the skin.
• the invention relates more particularly still mixtures or cocktails of different HPV DNAs (or probes containing these HPV DNAs or sequences thereof), which can be used in combination to carry out global diagnostics of the different forms of papillomavirus infections, possibly for the purpose of prognosis possible course of infection.
• Preferred mixtures in accordance with the invention are identified in Table V which follows.
• each of these mixtures can also be defined as comprising at least one of the new probes according to the invention.
• the diagnostic compositions according to the invention can be defined as containing:
• the different mixtures distinguish between infections with a low risk of cancerization and infections with a high risk of cancerization.
• hybridization of a viral preparation from a patient under diagnosis with mixture 7 will show a greater risk of skin cancerization than in the case where hybridization will occur more with mixture 3.
• the EVs detected by the mixture 5 will testify to a risk of. greater cancerization than the EVs detected by the mixture 6.
• the mixture 4 will detect EVs at even higher risk than those detected by the mixture 5.
• Other mixtures or cocktails of different DNA-HPVs are further described below. probes containing these DNA-HPVs or sequences thereof), which can be used in combination to carry out global diagnostics of the various forms of papillomavirus infections, possibly for the purpose of predicting the possible course of the infection.
• HPV-IP2 can be considered to be particularly representative of probes usable for the detection of the risks of development of genital neoplasias and, in particular, of cancers of the cervix.
• the invention therefore more particularly still relates to diagnostic kits or "kits” comprising at least 10 groups appearing in groups numbered from 1 to 10 in the table under the heading "Designation of mixtures”.
• the basic principle of in vitro detections of DNAHPV will naturally involve hybridizations carried out under strict or less strict conditions.
• the object of examinations involving probes prepared from mixtures of DNAs of cloned HPVs is to demonstrate an HPV and to identify the type of HPV in a biopsy, in cells obtained by scraping lesions, or in biopsy sections fixed by the Carnoy mixture (ethanol, chloroform, acetic acid 6: 3: 1) and included in the paraffin.
• the examination requires the prior extraction of DNA from the samples according to methods of which the principle is known and involves the analysis of this DNA by molecular hybridization experiments, carried out under strict or less strict conditions, to using radioactive probes (labeled with 32 P or S) prepared from DNA mixtures of HPVs. Each test usually requires the use of more than one probe mix.
• hybridization methods can be used.
• radioactive hybrids are detected after exposure of the membranes in contact with an X-ray film.
• the radioactive probes consist either of HPV DNAs labeled by the "nick" method translation ", or by RNAs prepared by transcription of viral DNAs inserted into a vector, for example of the SP6 type.
• the use of radioactive probes has the advantage of great sensitivity, but this does not exclude the use non-radioactive probes, for example biotinylated and capable of being recognized by antibodies either labeled themselves, or themselves recognized by antibodies carrying an enzymatic, fluorescent marker, etc.
• the choice of probes depends on the nature of the samples. Thus, for example, in the case of a patient suspected of having EV, mixtures 1, 2, 3, 4, 5, 6 and 7 will be used.
• Mixtures 1 and 2 will make it possible to make the differential diagnosis between EV and skin warts.
• Probe 3 including the most frequently detected member of each of the three groups of HPVs associated with the disease, and probe 7, containing the DNAs of the types of HPV associated with cancer.
• EV will allow the diagnosis of he majority of EVs and, in particular, to identify patients infected with the types of HPVs presenting a risk for the development of cancers.
• the use of mixtures 4, 5 and 6 will make it possible to specify the type or types of HPV infecting the same patient.
• kits or "kits” containing a plurality of the above-indicated probes, in particular: either representatives of each of the 19 types and subtypes of DNA-HPVs mentioned above, - or mixtures of probes, preferably the various groups or mixtures of probes which have been defined above, these "kits” being intended for diagnostic studies "in vitro by hybridization between viral preparations obtained from patients and the various groups or mixtures.
• the invention is in no way limited to those of its modes of application and embodiments which have been more especially envisaged; on the contrary, it embraces all its variants; in particular the reference in the claims to a DNA-HPV designation followed by a determined number, and to which corresponds a DNA-HPV whose restriction map has been provided in the drawings, is understood to mean that these claims cover all the DNA-HPVs which have in common with this particular DNA-HPV can be classified in the same type, according to the definition which was given above, and a fortiori to DNA-HPV belonging to the same subtype.
• the invention relates more particularly to the products of expression of the E6, E7 and especially L2 genes, of the various papillomaviruses which have been mentioned in the above.
• These expression products can themselves be used for the detection of papillomaviruses or their expression products in determined biological samples and for the identification of papillomaviruses according to the types or subtypes to which they may belong.
• the conditions under which these expression products can be obtained will be illustrated in the rest of this description, in particular in relation to the expression of the L2 sequence of the HPV la papillomavirus, it being understood that similar techniques can be used to induce expression of L2 (or E6, E7 or L1) genetic sequences from other types of papillomavirus.
• L2 sequences or genes there will be, with some exceptions, more particularly reference to L2 sequences or genes, it being understood however that the lessons provided with regard to the expression products of the L2 genes may possibly be transposed to the expression products of the other genes of which question above.
• the products of expression of the L2 sequences are however of very particular interest, in that they can themselves be used for the production in vivo of antibodies capable of recognizing the products of expression of the L2 gene in samples.
• the new embodiment of the invention which is discussed below therefore provides separate means for determining the types of papillomavirus present in a lesion studied, in order to assess its severity and to choose the therapy to be followed.
• the antibodies produced against the various types of expression products of the L2 genes can be grouped in mixtures corresponding to those which have been identified above in connection with the hybridization probes.
• kits or kits comprising a plurality of distinct antibodies and allowing successive tests for detection, if necessary of identification or classification, of newly isolated papillomaviruses.
• a kit or kit in accordance with the invention comprises a plurality of reagents made up of antibodies or mixtures of distinct antibodies, for example reagents comprising respectively antibodies formed against the expression products of the grouped papillomavirus L2 genes as follows: 1) at least HPV2d,
• the antibodies formed against the expression products of the L2 genes can be used for the direct detection of viruses in histopathological sections from lesions induced by papillomaviruses in those affected.
• the detection is done on preparations fixed beforehand under dissociating conditions, for example with the medium or mixture of CARNOY already mentioned above (also described in the work by L. LISON, entitled (Histochemistry and cytochemistry of animals ”) .
• the anti-L2 antibodies which may be attached can be recognized by other antibodies formed against the former, these other antibodies carrying appropriate markers, preferably non-radioactive. These markers are for example of an enzymatic or fluorescent nature.
• the invention naturally also relates to the polypeptides themselves which result from the expression of the L2 genes of the papillomaviruses. These expression products have already in the above been called "L2 proteins". It also relates to the polypeptides in which this L2 protein is fused with other polypeptide sequences, insofar as these do not essentially modify the immunogenic properties of the L2 protein. The presence of these others Polypeptide fragments can in particular result from the mode of production used for these hybrid polypeptides, especially when they have been obtained by processes involving techniques of genetic engineering.
• the invention relates to hybrid polypeptides containing a sequence derived from beta-galactosidase. Such products can in particular be obtained by transformation of E.
• coli with appropriate vectors (phages or plasmids) modified by all or part of the lactose operon and comprising, in addition, inserted downstream of the promoter of the lactose operon (or any other suitable promoter, for example phage lambda), the nucleotide sequence derived from an L2 gene derived from a papillomavirus of determined type.
• phages or plasmids modified by all or part of the lactose operon and comprising, in addition, inserted downstream of the promoter of the lactose operon (or any other suitable promoter, for example phage lambda), the nucleotide sequence derived from an L2 gene derived from a papillomavirus of determined type.
• plasmids or phages of this type comprising at least part of the beta-galactosidase gene of the lacton operon.
• the invention also relates to groups of distinct polypeptides, these polypeptides each corresponding to only part of the complete L2 proteins mentioned above, it being understood, however, that the polypeptides of these various groups each include the antigenic sites characteristic of the L2 proteins of the genus in question.
• the polypeptides according to the invention can also, when they have been purified, be used in techniques for purifying the antibodies which correspond to them, in particular from sera from animals which had been immunized with these polypeptides.
• these polypeptides can be attached to affinity columns.
• the antibody purification operations then consist in passing the serum containing them in contact with affinity columns carrying the above-mentioned polypeptides.
• Antibodies selectively attached to these columns can then be recovered by dissociation of the antigen-antibody complexes, using an appropriate buffer, having an adequate ionic strength, for example a solution of a salt such as ammonium acetate. Acidified solutions can also be used.
• an appropriate buffer having an adequate ionic strength, for example a solution of a salt such as ammonium acetate. Acidified solutions can also be used.
• the invention relates to the compositions involving these antigens (or groups of antigens) and antibodies (or groups of antibodies).
• the invention relates to groups containing one or, preferably, a "cocktail" of antibodies derived from papillomavirus sets, all of which are said to be often present in a given type of condition.
• These cocktails containing these antibodies or groups of antibodies: mixture of sera or antibody compositions purified in association with an appropriate pharmaceutical vehicle
• These sera are then likely to cause a regression of the infections induced by papillomaviruses of the corresponding types or subtypes.
• the invention relates to the corresponding vaccinating compositions containing one or preferably several L2 proteins, in combination with a pharmaceutically acceptable vehicle suitable for the chosen mode of administration, in particular by parenteral route which can be used to protect people subjected to high risks of being affected by affection corresponding.
• fragment used of the open phase of L2 must each time be fused in phase in the vector used, where appropriate with a gene coding for a protein ensuring the stability or facilitating the subsequent purification of the hybrid protein then formed.
• the plasmid DNAs were obtained from pHPVI.a (Danos et al, 1982 and French patent 82 05887 of April 5, 1982) contained in E. coli (C 600 cells), of pCQV2 which housed RRI cells (queen, 1983) and pMC1403 which hosted MC1000 cells (Casadaban, 1983).
• the intermediate plasmids constructed and the plasmid pHPL2 were passed several times into E. coli MM 294 cells and the plasmids pHPL2-beta-galactosidase into MC1000 cells deficient in beta-galactosidase. Lac strains were detected as red colonies on Me Conkey's agar plates containing lactose (Silhavy et al, 1984, in "Experiments with gene fusions" - Cold Spring Harbor Laboratory , Cold Spring Harbor, NEW-YORK).
• the plasmids pHPL2 and pHPL2-beta-gal were then transfected into a proton deficient Ion strain, CAG1139 (Grossman et al, 1983, Cell 3 2, 151159).
• pHPL2 or pHPL2-beta-gal 116 were diluted 5 to 20 times in LB medium. They were then cultivated at 30oC, until reaching an Optical Density 600 of 0.5-0.9. The lambda-PR promoter was then depressed by growing the cells at 41oC for 90 minutes. The cultures were recentrifuged and the pellets collected, suspended in a 62 mM Tris medium, pH 6.8 containing 2% SDS, 26% glycerol, 2 M mercaptoethanol and 0.03% bromophenol blue. A CAG 1139 cell lysate containing no plasmid was used as a control.
• the protein bands were visualized by coloring with the dye known as "amido black” and transferred to a nitrocellulose sheet for analysis by the method known as “western blot” ("Western transfer”).
• the culture was then quickly brought to 41oC and cultivated at high temperature for 90 minutes.
• the cell pellet was resuspended in 20 mM Tris medium, pH 7.5, Mg Cl 2 10 mM, and subjected to a cell rupture treatment with ultrasound.
• the fusion protein soluble in the preparation was purified by affinity chromatography on a p-aminophenyl- ⁇ -D-thiogalactoside (TPEG) column ⁇
• the purified fusion protein as indicated above was used to immunize guinea pigs
• the L2 open reading frame was cloned for expression in Escherichia coli (E. coli).
• the plasmid pHPVl.a contains the entire genome of HPVl.a cloned at the BamHI site of the plasmid pBR322 by Danos et al (EMBO Jl, p. 231-236, 1982).
• a Hind III-Hpa II fragment from a plasmid pHPV Ia was subcloned into a plasmid pBR322.
• the plasmid obtained was called pHPL9.
• This plasmid was shortened by removing a non BamHI-PVu II fragment essential in the pBR322 region. From the resulting plasmid pHPL 9.3, an XhoII-Xmnl fragment was isolated comprising the 1526 base pairs (bp) of the open reading frame minus the 318 5 'terminal pairs. The stop codon of the open reading frame L2 is retained.
• the L2 fragment obtained was then inserted into the expression vector pCQV2 (queen 1983) in place of a non-essential BamHI-PvuII fragment.
• the open reading frame L2 is thus directly adjacent to the starting ATG codon and to the SD sequence (Shine and Dalgarno, Proc. Natl. Acad. Sci. USA 71, p. 1342-1346, 1974) of the phage cro gene lambda and under the control of the lambda PR promoter.
• the promoter is regulated by the temperature sensitive repressor CI857, allowing it to control the production of the L2 expression product. Plasmid pHPL2 was thus obtained.
• This DNA which contains the L2 gene and the sequences providing the transcription and translation signals, was shortened by digestion with PstI, before being inserted into the plasmid pMC1403 (Casadaban, Methods in enzymology 100, p. 293-308 , 1983) instead of Pstl-Smal fragment.
• PMC1403 contains the lac operon without its promoter and the first 22 base pairs of the open phase of the beta-galactosidase gene.
• the recombinant was transfected into MC 1000, lac- cells.
• the production at high temperature of beta-galactosidase in the clones obtained made it possible to select on Me Conkey lactose agar plates (Silhavez et al, Cold Spring Harbor Laboratory, NY 1984) the plasmids pHPL2 beta beta in which the genes for L2 and beta-galactosidase are in phase.
• DNA sequencing indicated that only the last two C-terminal amino acids of the open reading frame L2 had been lost during digestion by Bal 31 and that the product expressed by the open reading L2 was linked by a proline to the ninth amino acid of beta-galactosidase.
• PRODUCTION OF HPVl RELATED PROTEINS a BY CELLS TRANSFECTED WITH PHPL2 and PHPL2. beta gal 116.
• the plasmid pHPL2 has a coding capacity for an L2 protein of approximately 51.2 Kd and of pHPL2-beta beta for a hybrid protein of approximately 167 Kd.
• the proteins related to the HPVl.a viruses were revealed by analysis according to the so-called technique of "Western Blot", with anti-virus antibodies HPVl.a.
• the analysis results were the same for cells containing or not containing a plasmid, but for cells induced at 41 ° C, specific protein bands were demonstrated in the transfected cells: among proteins isolated from cells transfected with the recombinant pHPL2-beta 116, a main band having the expected molecular weight, of approximately 167 Kd, was isolated, corresponding to a fusion protein L2 beta-galactosidase (L2-beta gay ), and several minor bands of molecular weight of about 58 Kd, probably resulting from proteolytic degradation.
• pHPL2 a protein band of approximately 72 Kd was revealed. This molecular weight is higher than that expected of 51.2 Kd. The significance of this difference remains to be elucidated.
• the gay L2-beta product of the bacterial lysate of a heat-induced culture was purified, by chromatographic affinity on a column of TPEG-SEPHAROSIS (Ullman, Gene 29, pp. 27-31, 1984). Proteins eluted from the column were analyzed on a polyacrylamide-SDS gel. Staining with an "amido-black" revealed three main protein bands, one of high molecular weight probably corresponding to the hybrid protein L2-beta-gay, a second migrating jointly with the beta-galactosidase and a third with a molecular weight of about 60 Kd.
• the sera recognized proteins (approximately 60 and 55 Kd) also found in the CAG 1139 control lysates. At least one of them appears to correspond to the protein copurified with the protein L2-beta fusion cells and beta galactosidase.
• the sera recognized proteins of approximately
• the guinea pig sera obtained did not precipitate the intact viral HPVl.a particles used as antigens, which again indicated that the viral antigens recognized by the serum in the immunoblot technique may not be available on the surface of the virion or that the sera do not precipitate.
• type antigens characterized by the absence of cross-antigenic reaction between the different kinds of papillomaviruses, using antibodies obtained after injection of whole virions, - and group antigens, mostly masked in the virion, and demonstrated by antibodies after injection of dissociated viral particles.
• the papillomaviruses belonging to different types in the antibody-antigen reaction tests are those whose L2 coding sequences, preferably devoid of their respective homologous regions, code for polypeptides which do not allow the performance of antigen-antibody cross reactions with their respective antibodies.
• Antigens of preferred types are those which are encoded by the open reading frames of the L2 gene lacking the N-terminal region, in particular over 1/4 of the length of the L2 regions in question.
• the invention also relates to a test for detecting, in viruses belonging to different types, those of the polypeptides coded by the L2 regions which do not give cross-reactions.
• These polypeptides can be defined as those whose antibodies react effectively with viruses. contained in histological or cytological sections. fixed by the middle of Carnoy, but which react only badly, if not even at all, with the same cuts when these are fixed by the middle of Bouin.
• the invention also relates to a method for classifying new papillomaviruses against known papillomaviruses. This process is defined in claim 8 which also forms part of the present description.
• the invention also relates to the diagnostic method comprising the identification of the type of infectious virus possibly present in a patient, consisting in reacting antigens previously obtained against viruses belonging to different types with a biological sample, in particular of serum, from a patient for whom the test should be realized .
• infectious virus will be presumed to belong to a determined type as soon as an antigen-antibody reaction is observed between a serum sample and an antigen originating from a papillomavirus belonging to this same type.
• diagnostic tests can be carried out for example by implementing the ELISA method.
• the invention relates to a method for detecting whether or not an infectious papillomavirus belongs in a human biological sample, such as a tissue or fluid, for example a serum, to a given type of papillomavirus.
• a human biological sample such as a tissue or fluid, for example a serum
• This method is characterized by bringing this biological sample into contact with antibodies previously formed against a product of expression of a DNA containing at least part of the L2 region of the genome of a virus belonging to this type, this setting in contact being carried out under conditions and for a period allowing the carrying out of an immunological reaction.
• the detection of the formation of an antigen-antibody complex then testifies to the presence of a papillomavirus having a type identical or related to that from which the abovementioned expression product originated.
• the DNA containing the L2 region or the corresponding part has been cloned into a competent cellular host, such as a bacterium, for example E. coli.
• a competent cellular host such as a bacterium, for example E. coli.
• the part of the L2 region used corresponds to the open reading frame of the L2 gene devoid of the N-terminal region which is not characteristic of type.
• this N-terminal region corresponds to the first quarter of this reading frame.
• the invention relates to a such a detection method in which the DNA containing the above-mentioned part of the L2 region (or the entire L2 region) is a hybrid DNA formed from a nucleic acid coding for a protein normally expressible in the chosen competent cellular host and in which the aforementioned L2 region will have been incorporated beforehand, in particular by in vitro recombination.
• the protein normally expressible in the cell host corresponds to all or part of the beta-galactosidase, when this cell host is E. coli.
• the detection method according to the invention is applicable to any fixed histological section, preferably in the Carnoy medium, or even directly to a serum.
• the method according to the invention also comprises bringing the biological sample into contact under the conditions indicated above with "cocktails" of antibodies previously formed against products of expression of the L2 regions - or of their respective corresponding parts - originating from several viruses of the same or related types, in particular from viruses grouped in the same manner as indicated above, in connection with DNA probes.
• the invention finally relates to a method of manufacturing each of the above-mentioned hybrid polypeptides and corresponding antibodies.
• This process includes: the incorporation, in particular in vitro, of the L2 region of the genome of the papillomavirus concerned or of the corresponding region part in an appropriate vector;
• the invention also relates to the method for producing said antibodies, characterized by immunizing an animal, for example, a rabbit, with the above-mentioned polypeptides, and recovering the antibodies formed.
• the invention optionally includes the grouping either of hybrid peptides or of antibodies, obtained from various papillomaviruses, according to the different types to which these may prove to belong.

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